Somatic mutations of the HER2 kinase domain in lung adenocarcinomas

Mutations in the epidermal growth factor receptor gene (EGFR) in lung cancers predict for sensitivity to EGFR kinase inhibitors. HER2 (also known as NEU, EGFR2, or ERBB2) is a member of the EGFR family of receptor tyrosine kinases and plays important roles in the pathogenesis of certain human cancers, and mutations have recently been reported in lung cancers. We sequenced the tyrosine kinase domain of HER2 in 671 primary non-small cell lung cancers (NSCLC), 80 NSCLC cell lines, and 55 SCLCs and other neuroendocrine lung tumors as well as 85 other epithelial cancers (breast, bladder, prostate, and colorectal cancers) and compared the mutational status with clinicopathologic features and the presence of EGFR or KRAS mutations. HER2 mutations were present in 1.6% (11 of 671) of NSCLC and were absent in other types of cancers. Only one adenocarcinoma cell line (NCI-H1781) had a mutation. All HER2 mutations were in-frame insertions in exon 20 and target the identical corresponding region as did EGFR insertions. HER2 mutations were significantly more frequent in never smokers (3.2%, 8 of 248; P=0.02) and adenocarcinoma histology (2.8%, 11 of 394; P=0.003). In 394 adenocarcinoma cases, HER2 mutations preferentially targeted Oriental ethnicity (3.9%) compared with other ethnicities (0.7%), female gender (3.6%) compared with male gender (1.9%) and never smokers (4.1%) compared with smokers (1.4%). Mutations in EGFR, HER2, and KRAS genes were never present together in individual tumors and cell lines. The remarkable similarities of mutations in EGFR and HER2 genes involving tumor type and subtype, mutation type, gene location, and specific patient subpopulations targeted are unprecedented and suggest similar etiologic factors. EGFR, HER2, and KRAS mutations are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers.     

Somatic mutations of the ERBB4 kinase domain in human cancers

The EGFR family consists of 4 receptor tyrosine kinases, EGFR (ERBB1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4). Recent reports revealed that the kinase domains of both EGFR (ERBB1) and ERBB2 gene were somatically mutated in human cancers, raising the possibility that the other ERBB members possess somatic mutations in human cancers. Here, we performed mutational analysis of the ERBB4 kinase domain by polymerase chain reaction-single-strand conformation polymorphism assay in 595 cancer tissues from stomach, lung, colon and breast. We detected the ERBB4 somatic mutations in 3 of 180 gastric carcinomas (1.7%), 3 of 104 colorectal carcinomas (2.9%), 5 of 217 nonsmall cell lung cancers (2.3%) and 1 of 94 breast carcinomas (1.1%). The 12 ERBB4 mutations consisted of 1 in-frame duplication mutation and 8 missense mutations in the exons, and 3 mutations in the introns. We simultaneously analyzed the somatic mutations of EGFR, ERBB2, K-RAS, PIK3CA and BRAF genes in the 12 samples with the ERBB4 mutations and found that 1 gastric carcinoma with ERBB4 mutation also harbored K-RAS gene mutation. Our study demonstrated that in addition to EGFR and ERBB2, somatic mutation of the kinase domain of ERBB4 occurs in the common human cancers, and suggested that alterations of ERBB4-mediated signaling pathway by ERBB4 mutations may contribute to the development of human cancers.     

Comparative analysis of p53 gene mutations and protein accumulation in human non-small-cell lung cancer

A series of 54 resected primary non-small-cell lung carcinomas was analyzed for p53 gene mutations and for p53 protein accumulation and the findings were correlated with clinical parameters. Mutations in exons 5 through 8 of the p53 gene were identified by a denaturing gradient gel electrophoresis (DGGE) assay and cycle sequencing, whereas p53 protein accumulation was detected in paraffin-embedded tissue by immunostaining using 2 different murine monoclonal antibodies (MAbs) (BP53-12 and DO7). A p53 gene mutation and/or p53 protein accumulation was found in 37 of 54 tumors. Mis-sense mutations were closely associated with positive immunostaining, which was intense in 15 out of 17 cases with a mutation. In 10 tumors, obvious p53 accumulation was detected in the absence of mutations in exons 5 through 8. Conversely, only one of 8 p53 non-sense mutations led to detectable p53 accumulation. The most frequent single base changes were G → T transversions and C → T transitions. The presence of a p53 alteration was not related to age, tumor size, stage or histology. However, we found a significant inverse correlation between p53 alterations and the presence of a K-ras mutation. This was reflected in the overall postoperative survival data: patients with p53 alterations in their tumors tended to have a better prognosis than those without a p53 alteration; however, this difference was lost when cases with a K-ras mutation were omitted from the analysis. © 1995 Wiley-Liss, Inc.     

线粒体DNA突变与乳腺癌相关性的研究进展

乳腺癌体细胞线粒体DNA（mtDNA）编码区和调控区会发生多种突变,大量研究提示mtDNA突变积累可能是引起线粒体功能持久缺陷的关键因子,进而导致乳腺癌的发生与进展。本综述归纳了乳腺癌mtDNA点突变、缺失及其拷贝数改变在乳腺癌发生发展、病理生理过程中的作用,讨论了“线粒体——细胞核逆行响应”信号通路及mtDNA作为新的肿瘤标志物和治疗靶标的潜在临床价值。     

Inactivating mutations targeting the chfr mitotic checkpoint gene in human lung cancer

A hallmark of cancer is inactivation of cell cycle checkpoints. However, very few mutations targeting mitotic checkpoint genes have been described, and in those instances, a wild-type copy of the gene was retained. chfr is a mitotic checkpoint gene that functions in early prophase delaying chromosome condensation in response to microtubule poisons. In a panel of 53 lung carcinomas for which matched normal tissue was available, we identified three missense mutations in the chfr gene, at least one of which was associated with loss of heterozygosity. In tissue culture checkpoint assays, the tumor-associated missense mutants had reduced activity or were inactive. Together with recent data suggesting that the chfr gene is frequently silenced in various tumors because of methylation of its promoter, these findings suggest that chfr is inactivated by multiple mechanisms in human cancer.     

The ras gene family in human non-small-cell lung cancer.

The three ras genes code for proteins with a putative role in cellular signal transduction. They belong to a larger family of small guanosine-triphosphate (GTP)-binding proteins. The ras proteins acquire transforming activity when amino acids are substituted at one of a few specific sites, as a result of a point mutation in the gene. In about one third of adenocarcinomas of the lung, a K-ras mutation is present in codon 12 of the gene. Patients with early stages of K-ras mutation-positive tumors have a very unfavorable prognosis, even if apparently radical resection of the tumor has taken place. K-ras mutations are very rare among nonsmokers, and it is reasonable to assume that carcinogens in tobacco smoke directly cause the mutation. The types of ras mutations found in lung cancer are different from those in gastrointestinal malignancies. Colon cancer is mainly associated with mutations leading to substitution of the normal glycine at amino acid position 12 of K-ras by either valine or aspartic acid, and mutations in N-ras are not exceptional. In contrast, the predominant mutation in lung cancer leads to substitution of cysteine in codon 12. Several other members of the ras gene superfamily are also expressed in human lung cancer, but a possible relationship with lung tumorigenesis remains to be established.     

Non-small-cell lung cancer harbouring mutations in the EGFR kinase domain

Key “driver” mutations have been discovered in specific subgroups of non-small-cell lung cancer (NSCLC) patients. Activating mutations in the form of deletions in exon 19 (del 19) or the missense mutation L858R in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) predict outcome to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Pooled data from several phase II studies show that gefitinib and erlotinib induce responses in over 70% of NSCLC patients harbouring EGFR mutations, with progression-free survival (PFS) ranging from 9 to 13 months and median survival of around 23 months. Two studies in Caucasian and Asian patients have confirmed that these subgroups of patients attain response rates of 70% with erlotinib and gefitinib, including complete responses, PFS up to 14 months and median survival up to 27 months. These landmark outcomes have been accompanied by new challenges: the additional role of chemotherapy and the management of tumours with the secondary T790M mutation that confers resistance to EGFR TKIs. Mechanisms of resistance to reversible EGFR TKIs should be further clarified and could be related to modifications in DNA repair. The presence of double mutations (T790M plus either L858R or del 19) at the time of diagnosis could be much more frequent than originally thought. The sensitivity to EGFR TKIs could be greatly influenced by the expression of genes involved in the repair of DNA double-strand breaks by homologous recombination and non-homologous end joining.     

Somatic deletions and mutations of c-Ha-ras gene in human cervical cancers

The c-Ha-ras-1 locus was analysed in cervical cancers and shown to exhibit the loss of one allele in 36% of heterozygous tumours and a mutation at codon 12 in 24% of tumours at advanced stages. 40% of tumours with mutation contained also a deletion. A c-myc gene activation was found in 100% and 70% of tumours containing mutation and deletion respectively. This suggests that the two proto-oncogenes cooperate for the progression of cervical cancers. Furthermore as more than 90% of these tumours contained also human papillomavirus sequences, our data strongly suggest that multiple genetic events are involved in the genesis and progression of most cervical cancers.     

Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.     

Missense mutations of the BRAF gene in human lung adenocarcinoma

Mutations of the BRAF protein serine/threonine kinase gene have recently been identified in a variety of human cancers, most notably melanomas. We sought to determine the frequency of BRAF mutations in human lung cancer pathogenesis. Analysis of BRAF sequence from 127 primary human lung adenocarcinomas revealed mutations in two tumor specimens, one in exon 11 (G465V), and a second in exon 15 (L596R). These specimens belong to the same adenocarcinoma subgroup as defined by clustering of gene expression data. BRAF may provide a target for anticancer chemotherapy in a subset of lung adenocarcinoma patients.     

Somatic mutations in the BRCA1 gene in Chinese women with sporadic breast cancer

Recent studies suggested that breast cancer patients who carry a BRCA1 germline mutation benefit from poly (ADP-ribose) polymerase (PARP) inhibitors; therefore, it would be of great interest to detect BRCA1 somatic mutations in sporadic breast cancers. In this study, we detected BRCA1 somatic mutations in tumor cDNA from 144 Chinese women with sporadic breast cancer by using polymerase chain reaction (PCR)-direct sequencing assay. In total, eight BRCA1 alterations (three nonsense mutations and five missense mutations) were identified in this cohort of 144 sporadic breast cancers. We further confirmed that 5 out of 144(3.5%) sporadic breast cancer cases carried a BRCA1 somatic mutation, including two novel nonsense mutations (c.191_212del22 and c.2963C>G) resulting in a truncated protein and three missense mutations (c.114G>T, c.925A>C, and c.824G>A). The two cases with BRCA1 somatic truncating mutations also contained a TP53 somatic mutation in the tumors. Our study suggested that a small subset of sporadic breast cancers do harbor BRCA1 somatic mutations; these patients who carry a BRCA1 somatic mutation may be potential candidates for treatment with PARP inhibitors.     

The protein tyrosine kinase family of the human genome

As the sequencing of the human genome is completed by the Human Genome Project, the analysis of this rich source of information will illuminate many areas in medicine and biology. The protein tyrosine kinases are a large multigene family with particular relevance to many human diseases, including cancer. A search of the human genome for tyrosine kinase coding elements identified several novel genes and enabled the creation of a nonredundant catalog of tyrosine kinase genes. Ninety unique kinase genes can be identified in the human genome, along with five pseudogenes. Of the 90 tyrosine kinases, 58 are receptor type, distributed into 20 subfamilies. The 32 nonreceptor tyrosine kinases can be placed in 10 subfamilies. Additionally, mouse orthologs can be identified for nearly all the human tyrosine kinases. The completion of the human tyrosine kinase family tree provides a framework for further advances in biomedical science.     

肝癌患者β-catenin基因第三外显子的体细胞突变

目的探讨中国地区人肝癌发生发展过程中有无β-catenin基因突变.方法采用PCR-SSCP方法,设计扩增β-catenin基因第三外显子的引物,检测20例肝癌患者及7株人肝癌细胞中有无β-catenin基因突变,并通过DNA测序及限制性内切酶对突变加以验证.结果20例肝癌患者中有2例发生β-catenin基因突变,均为41位密码子由编码苏氨酸变成编码丙氨酸.7株人肝癌细胞均无β-catenin基因突变.结论10%(2/20)的肝癌组织样品有β-catenin基因突变.提示β-catenin基因突变可能参与了部分肝癌癌变过程,但不是中国地区肝癌发生发展过程中的主要和关键原因.     

ras gene mutations in human prostate cancer

Point mutations at codons 12, 13, or 61 of the Ha-, Ki-, and N-ras genes are able to convert these normal cellular genes into activated oncogenes. Previous studies have shown that ras gene mutations occur in a variety of human solid tumors and may be important in the pathogenesis of some of these tumors. In order to test the hypothesis that ras gene mutations may be associated with prostate cancer, we have used an oligodeoxynucleotide hybridization assay to detect wild-type and mutant alleles in genomic DNA from prostate tumors and prostate tumor cell lines amplified using the polymerase chain reaction. Twenty-four primary prostate tumors (23 acinar tumors and one ductal tumor) and five prostate tumor cell lines were examined for mutations at codons 12, 13, and 61 of the Ki-ras, Ha-ras, and N-ras genes. Two mutations were detected: an A----G transition causing a glutamine to arginine amino acid substitution at codon 61 of the Ha-ras gene in a primary prostatic duct adenocarcinoma and a G----T transversion causing a glycine to valine amino acid substitution at codon 12 of the Ha-ras gene in a prostate tumor cell line (TSU-PR1) derived from a lymph node metastasis. While the overall frequency of ras gene mutations in prostate tumors is low, when these mutations do occur they may have a role in the progression of disease or the development of the unusual ductal variant of prostatic adenocarcinoma.     

Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer.

Inherited mutations in the BRCA1 gene confer increased susceptibility to breast and ovarian cancer. Its role in sporadic carcinogenesis is not well defined. Somatic mutations in breast cancers have not been reported and to date there are only three reports of somatic mutations in sporadic ovarian cancers. To investigate the contribution of BRCA1 mutations to sporadic breast and ovarian cancer in the Chinese population, we analysed 62 samples from Chinese women using the protein truncation test. There were 40 cases of breast cancer under age 50 and 22 cases of ovarian cancer, all unselected for family history. There was no age selection for the ovarian cancers. We found two somatic BRCA1 mutations in exon 11, one in a breast cancer and the other in an ovarian cancer, both of which result in truncated proteins. Our results indicate that somatic BRCA1 mutations, like somatic mutations in the BRCA2 gene, though very rare, can be found in both breast and ovarian cancers and support a tumor suppressor function for BRCA1 in sporadic tumors.     

Atypical protein kinase C iota expression and aurothiomalate sensitivity in human lung cancer cells

The antirheumatoid agent aurothiomalate (ATM) is a potent inhibitor of oncogenic PKC iota. ATM inhibits non-small lung cancer (NSCLC) growth by binding PKC iota and blocking activation of a PKC iota-Par6-Rac1-Pak-Mek 1,2-Erk 1,2 signaling pathway. Here, we assessed the growth inhibitory activity of ATM in a panel of human cell lines representing major lung cancer subtypes. ATM inhibited anchorage-independent growth in all lines tested with IC(50)s ranging from approximately 300 nmol/L to >100 micromol/L. ATM sensitivity correlates positively with expression of PKC iota and Par6, but not with the PKC iota binding protein p62, or the proposed targets of ATM in rheumatoid arthritis (RA), thioredoxin reductase 1 or 2. PKC iota expression profiling revealed that a significant subset of primary NSCLC tumors express PKC iota at or above the level associated with ATM sensitivity. ATM sensitivity is not associated with general sensitivity to the cytotoxic agents cis-platin, placitaxel, and gemcitabine. ATM inhibits tumorigenicity of both sensitive and insensitive lung cell tumors in vivo at plasma drug concentrations achieved in RA patients undergoing ATM therapy. ATM inhibits Mek/Erk signaling and decreases proliferative index without effecting tumor apoptosis or vascularization in vivo. We conclude that ATM exhibits potent antitumor activity against major lung cancer subtypes, particularly tumor cells that express high levels of the ATM target PKC iota and Par6. Our results indicate that PKC iota expression profiling will be useful in identifying lung cancer patients most likely to respond to ATM therapy in an ongoing clinical trial.